I. A. MicroRNA profiling of the Bone Marrow Microenvironment, Serum and Plasma of Myeloma and Precursor Diseases. Multiple myeloma (MM) is consistently preceded by a precursor state (monoclonal gammopathy of undetermined significance, MGUS; or smoldering myeloma, SMM). We previously found that the extracellular bone marrow (BM) microenvironment in MM contains a unique microRNA (miR) signature, which is partially present in the peripheral blood serum (let-7a, let-7b, let-7i; miR-106b, miR-15a/b, miR-16, miR-20a, miR-21, miR-106b, and miR-361). A subset of these miRs showed aberrant expression in precursor disease MGUS, while aberrant expression of the remaining miRs were associated with disease progression and MM. During 2014 these findings were further validated in the plasma of a separate cohort of myeloma and healthy controls. Additionally, the miR profiles were assessed pre and post treatment to evaluate response to therapy. The findings obtained in 2014 are currently being incorporated into a revised manuscript as a validation study for our previous findings detailed in the 2013 Annual Report. During 2014 we also completed and published a revised manuscript with Dr. Ola Landgren detailing the results of our cytokine profiling study of the bone marrow microenvironment in myeloma. I. B. Monoclonal Gammopathy of Undetermined Significance (MGUS) and MicroRNAs in Ranch Hand Veterans. In 2014 we began and completed microRNA isolation from deidentified Air Force Heath Study (AFHS) serum samples received via our collaborators at the CDC/ATSD (Dr. Youn Shim and Dr. Robert Vogt). These samples were previously obtained at multiple timepoints, spanning three decades, from Air Force Ranch Hand Veterans exposed to TCDD. A subpopulation of the veterans ultimately developed MGUS, SMM, and/or MM. Dr. Ola Landgren's lab assayed the samples for SPEP, IFE, and serum free light chain ratio in 2013-2014. We are currently in the process of performing quantitative PCR on the RNA isolated from the serum samples, assaying a panel of miRs associated with MM, MGUS, SMM and TCDD exposure. In 2014-2015 miR data will be statistically analyzed for correlation with presence of M-protein levels over time, disease status, TCDD exposure, clinical outcome, demographic, and other available data. I. C. MicroRNA profiling of GATA2 deficiency associated myelodysplasia(MDS)/myeloid leukemia. Somatic and inherited germline mutations in GATA2 have been identified in patients diagnosed with monocytopenia, B-cell and NK-cell lymphopenia, susceptibility to opportunistic infections (e.g. disseminated MAC), and a strong propensity to develop hypocellular MDS/AML or CMML. In 2012 and 2013 we identified the apoptosis related gene MCL-1 as a potential target of miR-181c and GATA2 in cell lines derived from patients with GATA2 deficiency and MDS. Our studies indicate that MCL1 is significantly down-regulated in GATA2 deficiency patient derived cell lines. Deletion of Mcl1 is known to cause apoptosis and loss of HSCs in murine studies. The down-regulation of MCL1 may play a role in GATA2 deficiency by permitting apoptosis and the depletion of hematopoietic progenitors resulting in cytopenias, immunodeficiency and risk of MDS/AML. Studies in our lab suggest that the down-regulation of MCL1 in GATA2 deficiency may occur through a mechanism involving aberrant expression miR-181c. We continued functional studies in 2014 in cell lines to validate these findings. I.D. MicroRNA changes in chronic lymphocytic leukemia in response to treatment. In 2014 we began a collaboration with Dr. Adrian Wiestner in NHLBI to analyze microRNA levels in peripheral blood B-cells from patients pre and post treatment to correlate miR expression patterns within the B-cell signaling network in response to treatment with targeted inhibitors. This work is ongoing. II. A. Immunophenotypic and morphologic features distinguishing GATA2 deficiency associated bone marrow failure from idiopathic aplastic anemia. Germline GATA2 gene mutations, leading to haploinsufficiency have been identified in patients with familial myelodysplastic syndrome/ acute myeloid leukemia (MDS/AML), monocytopenia and mycobacterial infections (MonoMAC), Emberger syndrome, and dendritic cell, monocyte, B and NK-cell deficiency (DCML). GATA2 deficiency-associated bone marrow failure can present with features that overlap with idiopathic aplastic anemia (AA) and GATA2 mutations have been reported in a minority of patients with congenital neutropenia and AA. The bone marrow (BM) from patients with GATA2 deficiency is typically hypocellular with varying degrees of dysplasia. Distinguishing GATA2 patients from those with AA is critical for selecting appropriate therapy. We compared the BM flow cytometric, morphologic and cytogenetic features of 28 GATA2 patients to those of 32 patients being evaluated for idiopathic AA. The marrow of GATA2 patients had severely reduced monocytes, B-cells and NK-cells, absent hematogones, and inverted CD4:CD8 ratios. Atypical megakaryocytes and abnormal cytogenetics were more common in GATA2 marrows. CD34+ cells were comparably reduced in GATA2 and AA. Using these criteria, we prospectively identified 4 patients out of 32 with suspected AA who had features suspicious for GATA2 mutations, later confirmed by DNA sequencing. Our results show that routine BM flow cytometry, morphology and cytogenetics, in patients who present with suspected AA, can identify patients for whom GATA2 sequencing is indicated. A manuscript with the findings is currently in submission for publication. II.B. Immunophenotypic and morphologic analyses of other diseases with bone marrow and peripheral blood pathology were performed in 2014 as part of consultative collaborative research including Hairy cell leukemia, chronic lymphocytic leukemia, ADA2 deficiency, WHIM syndrome, aplastic anemia, PI3Kinase deficiency, and RAS-associated autoimmune leukoproliferative disorder.